| Assay Method Information | |
| | KRAS G12D Binding Assay |
| Description: | The binding ability of the compound to KRAS G12D protein was detected by TR-FRET method, and the inhibitory activity of the compound on KRAS G12D protein was evaluated. Human KRAS G12D His-tagged (Cisbio, 63ADK000CB27PEG) protein was 100-fold diluted with PPI Eu detection buffer (Cisbio, 61DB9RBF), then 5 μL of which was added to a 384-well plate and centrifuged at 2000 rpm for 40 seconds. A 10 mM concentrated stock solution of the test compound was prepared with dimethyl sulfoxide, 3-fold serially diluted, then diluted to make a working solution (0.5% DMSO) with PPI Eu detection buffer, added to a 384-well plate at 5 μL/well, with the control group added with 0.5% dimethyl sulfoxide and the highest concentration of the test compound at 10 μM, and centrifuged at 2000 rpm for 40 seconds. An 80 nM GDP standard working solution (Cisbio, 63ADK000CB27PEG) was prepared with PPI Eu detection buffer and added to a 384-well plate at 5 μL/well, with the final concentration of GDP standard working solution at 20 nM. The plate was centrifuged at 2000 rpm for 40 seconds and reacted at room temperature for 30 minutes. 1×6His Eu cryptate antibody and GTP-Red reagent (Cisbio, 63ADK000CB27PEG) were prepared with PPI Eu detection buffer, mixed in a volume ratio of 1:1, 5 μL of which was then added to a 384-well plate, centrifuged at 2000 rpm for 40 seconds, and incubated at room temperature for 30 minutes. |
| Affinity data for this assay | |
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